Current molecular biology practices require a significant amount of DNA cloning to provide necessary research tools. The motivation for developing a new system of precise gene transfer to generate these tools stems from separate factors. The first is that current methods regard in vitro manipulations that require undesirable quantities of DNA purification. For example, the ability to transfer a polynucleotide of interest to an expression vector is often limited by the availability or suitability of restriction enzyme recognition sites. Often, multiple restriction enzymes must be employed for the removal of the polynucleotide of interest, and the reaction conditions used for each enzyme may differ such that it is necessary to perform the excision reactions in separate steps. In addition, it may be necessary to remove a particular enzyme used in an initial restriction enzyme reaction prior to completing all restriction enzyme digestions, which requires a time-consuming purification of the subcloning intermediate. Ideal methods for the subcloning of DNA molecules would permit the rapid transfer of the target DNA molecule from one vector to another, preferably in vivo, without the need to rely upon restriction enzyme digestion(s). Furthermore, the larger the gene set, the greater the additional effort that is required.
Given that the need to manipulate large numbers of DNAs requires robotics that are frequently beyond the abilities of most small labs, a simpler way to transfer genes between different vectors, allowing smaller labs to efficiently study entire genomes at greatly reduced expense, is desirable.
U.S. Pat. No. 5,851,808 and Liu et al. (1998) are directed to the Echo™ Cloning system (also referred to as the Univector Plasmid Fusion System) (Invitrogen; Carlsbad, Calif.) that comprises a vector (pUNI) into which sequences encoding a gene of interest are inserted. The pUNI vector has a single sequence-specific recombinase target site, in specific embodiments a loxP site, preceding the insertion site for the gene of interest, a selectable marker gene and a conditional origin of replication that is active only in host cells expressing the requisite trans-acting replication factor. The pUNI vectors are designed to contain a gene of interest but lack a promoter for the expression of the gene of interest. Using a sequence-specific recombinase (e.g., Cre recombinase), a precise fusion of the pUNI vector into a second vector containing another copy of the sequence-specific recombinase target site as on the pUNI vector occurs. The second vector, referred to generically as a pHOST vector (or pAcceptor vector), is an expression vector that contains the sequence-specific recombinase target site downstream of the promoter element contained within the expression vector. Following the site-specific recombination event that occurs between the single sequence-specific recombinase target sites located on each vector (e.g., the pUNI vector and the pHOST vector), the two vectors are stably fused in a manner that places the expression of the gene of interest under the control of the promoter element contained within the expression vector. This fusion event also occurs in a manner that retains the proper translational reading frame of the gene of interest. This subcloning event occurs without the need to use restriction enzymes. The fusion or recombination event can be selected for by selecting for the ability of host cells, which do not express the trans-acting replication factor required for replication of the conditional origin contained on the pUNI vector, to acquire the selectable phenotype conferred by the selectable marker gene (if present) on the pUNI vector. The pUNI vector cannot replicate in cells lacking expression of the trans-acting replication factor and therefore, unless the pUNI vector has integrated into the second vector that contains a non-conditional origin of replication, pUNI will be lost from the host cell.
In technology similar to the Echo system, the site-specific recombination system of phage lambda, GATEWAY™ Cloning Technology (Invitrogen; Carlsbad, Calif.) (U.S. Pat. No. 6,277,608), allows transfer of DNA segments between different cloning vectors while maintaining orientation and reading frame, also effectively replacing the use of restriction endonucleases and ligase. Homologous recombination is not utilized. The phage lambda system utilizes the integration sites attB/attP. In specific embodiments, the method for cloning or subcloning desired nucleic acid molecules comprises a first step of combining in vitro or in vivo (1) one or more insert donor molecules comprising one or more nucleic acid segments flanked by two or more recombination sites, wherein the recombination sites do not substantially recombine with each other; (2) two or more different vector donor molecules, each comprising two or more recombination sites, wherein the recombination sites do not substantially recombine with each other; and (3) one or more site specific recombination proteins; and a second step of incubating the combination under conditions sufficient to transfer one or more of the nucleic acid segments into the different vector donor molecules, thereby producing two or more different product molecules.
U.S. Pat. No. 6,270,969 describes an in vitro method for apposing an expression signal and a gene or partial gene comprising (a) mixing in vitro a first nucleic acid molecule comprising an expression signal and at least a first recombination site, and a second nucleic acid molecule comprising a gene or partial gene and at least a second recombination site; and (b) incubating the mixture in vitro in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least the first and second recombination sites, thereby apposing the expression signal and the gene or partial gene such that expression of the gene or partial gene can be controlled by the expression signal.
Zhang et al. (2002) describes methods for recombination cloning that facilitate genomic library construction and screening. The technology is particularly useful for preparing gene-targeting constructs, such as for targeted gene disruption in mouse embryonic stem cells. Although multiple embodiments are addressed, a specific embodiment regarding utilization of targeting fragments generated in cells that are induced to express I-SceI and are simultaneously infected with library phage is described. The fragment that contains an antibiotic resistance marker for selection is released by digestion of the vector with I-SceI and then recombines with phage DNA carrying the proper homology. The amplified phage are collected and used to infect a cre-expression strain for automatic subcloning; these cells are then subjected to selection for identification of homologous recombinants. Survival occurs only for those cells having plasmids that have undergone recombination.
Despite these technologies available to a skilled artisan, there still remains the need for an in vivo method that does not require the need for DNA preparation of donor clones, is affordable even to small labs, provides flexibility for adaptor sequences, is compatible with other systems, and/or is less laborious than available methods. The present invention addresses these needs.